This application is in the area of pharmaceutical chemistry, and in particular includes sphingolipid derivatives, prodrugs and pharmaceutical compositions and salts thereof for the treatment of abnormal cell proliferation, including benign and malignant tumors, the promotion of cell differentiation, the induction of apoptosis, the inhibition of protein kinase C, and the treatment of inflammatory conditions, psoriasis, inflammatory bowel disease as well as proliferation of smooth muscle cells in the course of development of plaques in vascular tissue. The invention also includes a method for triggering the release of cytochrome c from mitochondria that includes administering an effective amount of a sphingolipid or its derivative or prodrug to a host in need thereof. Further, the invention provides a method for treating bacterial infections, including those that influence colon cancer and other disorders of the intestine, that includes administering an effective amount of one of the active compounds identified herein.
Sphingosine is the common name for D-erythro-4-trans-sphinganine, the prevalent long-chain base of most mammalian sphingolipids. It most often has 18 carbon atoms and the stereochemistry shown in FIG. 1. Sphingolipids generally are composed of a long-chain (sphingoid) base (sphingosine, sphinganine, 4-hydroxysphinganine, or a related compound) as the backbone moiety (Karlsson, K. A. Chem. Phys. Lipids, 5:6-43, 1970), which is usually modified by an amide-linked long-chain fatty acid (for ceramides), and a head group at position 1, as illustrated in FIG. 2. Over 300 classes of sphingolipids are known, most of which have head groups with simple to complex carbohydrates (see Merrill and Sweeley, New Comprehensive Biochemistry: Biochemistry of Lipids, Lipoproteins, and Membranes, (Vance, D. E. and Vance, J. E., eds.), pp. 309-338, Elsevier Science, Amsterdam, 1996). It is a common misconception from the names of these compounds (e.g., ceramide, sphingomyelin, gangliosides, etc.) that sphingolipids are only found in neuronal tissues. In fact, sphingolipids are major constituents of all eukaryotic (and some prokaryotic) organisms, including plants (Lynch, D. V., Lipid Metabolism in Plants (T. S. Moore, Jr., ed.), pp. 285-308, CRC Press, Boca Raton, Fla. 1993). This nomenclature merely reflects their initial discovery in brain tissues by classic studies a century ago (Thudichum, J. L. W., A Treatise on the Chemical Constitution of Brain, Bailliere, Tindall and Cox, (London) 1884).
Mammalian sphingolipid compounds typically vary in the presence or absence of: (I) the 4,5-trans-double bond (for example, sphingosine has a double bond whereas sphinganine (also referred to as dihydrosphingosine) does not); (ii) double bond(s) at other positions, such as position 8; (iii) a hydroxyl group at position 4 (D-1-hydroxysphinganine, also called xe2x80x9cphytosphingosinexe2x80x9d) or elsewhere (Robson et al., J. Lipid Res. 35:2060-2068, 1994); (iv) methyl group(s) on the alkyl side chain or on the amino group, such as N,N-dimethylsphingosine; and (v) acylation of the amino group (for example ceramide (also referred to as N-acylsphingosine), and dihydroceramide (also referred to as N-acyl-sphinganine)). The 4-hydroxysphinganines are the major long-chain bases of yeast (Wells, G. B. and Lester, R. L., J. Biol. Chem., 258:10200-10203, 1983), plants (Lynch, D. V., Lipid Metabolism in Plants (T. S. Moore, Jr., ed.), pp. 285-308, CRC Press, Boca Raton, Fla. 1993), and fungi (Merrill et al., Fungal Lipids (R. Prasad and M. Ghanoum, eds.), CRC Press, Boca Raton, Fla. 1995a), but are also made by mammals (Crossman and Hirschberg, J. Biol. Chem. 252:5815-5819, 1977). Other modifications of the long-chain base backbone include phosphorylation at the hydroxyl oxygen of carbon 1 (Buehrer and Bell, Adv. Lipid Res. 6:59-67, 1993), and acylation (Merrill and Wang, Methods Enzymol., 209:427-437, 1992) (Igarashi and Hakomori, Biochim. Biophys. Res. Commun. 164:1411-1416, 1989; Felding-Habermann et al., Biochemistry 29:6314-6322, 1990) of the amino group. Each of these compounds can be found in various alkyl chain lengths, with 18 carbons predominating in most sphingolipids, but other homologs can constitute a major portion of specific sphingolipid (as exemplified by the large amounts of C20 sphingosine in brain gangliosides) (Valsecchi et al., J. Neurochem., 60:193-196, 1993) and in different sources (e.g., C16 sphingosine is a substantial component of milk sphingomyelin) (Morrison, Biochim. Biophys. Acta., 176:537-546, 1969). One difficulty in studying these compounds is that relatively few are commercially available in chemically-pure form. For example, most of the sphinganine that can be purchased from various vendors is a mixture of the D and L enantiomers (therefore, commercially available dihydroceramides are also mixtures) and the metabolism, and some of the functions, of these compounds are sensitive to stereochemistry (Stroffel and Bister, Hoppe-Seyler""s Z. Physiol. Chem. 354:169-181, 1973; Buehrer and Bell, J. Biol. Chem. 267:3154-3159, 1992; Adv. Lipid Res. 26:59-67, 1993; Hauser et al., J. Biol. Chem. 269:6803-6809, 1994; Olivera et al., J. Biol. Chem. 269:17924-17930, 1994). For example, sphingosine kinase (which forms the signaling compound sphingosine 1-phosphate) will act only on the erythro stereoisomersxe2x80x94the threo stereoisomers are inhibitors of the enzyme (Buehrer and Bell, J. Biol. Chem. 267:3154-3159, 1992; Adv. Lipid Res. 26:59-67, 1993). Release of intracellular calcium is limited to D-(+)-erythrosphingosine 1-phosphate.
Sphingosines and other long-chain bases are cationic amphiphiles, which distinguishes them from most other naturally occurring lipids, which are neutral (including zwitterionic) or anionic. In the protonated form, they affect the phase behavior of both zwitterionic (Koiv et al., Chem. Phys. Lipids. 66:123-134, 1993; Lxc3x3pez-Garcia et al., Biochim. Biophys. Acta., 1194:281-288, 1994) and acidic (Koiv et al., Chem. Phys. Lipids. 66:123-134, 1993; Lxc3x3pez-Garcia et al., Biochim. Biophys. Acta. 1194:281-288, 1994) phospholipids.
The hydroxyl groups at positions 1, 3 and sometimes 4 or 6 are also relevant to the behavior of these compounds. This has mostly been considered from the perspective of how hydrogen bonding in the interfacial region of the bilayer affects membrane structure (Thompson and Tillack, Annu. Biophys. Chem., 14:361-386, 1985). However, in a study of phosphatidic acid phosphatase purified from yeast (Wu et al., J. Biol. Chem. 268:13830-13837, 1993), inhibition of this enzyme by long-chain bases showed a considerable preference for phytosphingosine and sphinganine over sphingosine, which matches the types of sphingoid bases found in yeast. Therefore, these functional groups appear to be present both for structural purposes and to allow optimum interaction with cellular targets.
Many microorganisms, microbial toxins and viruses bind to cells via sphingolipids. Specific organisms that have been reported as binding to sphingolipids include cholera toxin (ganglioside GM1) (Thompson, et al., Biochem. Pharmacol. 56:591-597, 1998); verotoxin (globosides) (Farkas-Himsley et al., Proc. Natl. Acad. Sci. (USA) 92:6996-7000, 1995; Bast et al., Infect. and Immun., 57:969-74, 1997); Shiga-like toxin 2e (globotriaosylceramide, Gb3) (Jacewicz et al., J. Clin. Invest. 96:1328-1335, 1995; Keusch et al., Infect. and Immun. 63: 1138-1141; 1995); and Clostridium botulinum type B neurotoxin (to synaptotagmin II associated with gangliosides GT1b/GD1a) (Nishiki et al., FEBS Lett. 378: 253-257, 1996).
Furthermore, many bacteria utilize sphingolipids to adhere to cell. Examples known in the art include Escherichia coli (galactosylceramide) (Blomberg et al., Infect. and Immun. 61: 2526-2531, 199; Payne et al., Infect. and Immun. 61: 3673-3677, 1993; Khan et al., Infect. and Immun. 64: 3688-3693, 1996); Haemophilus influenzae (gangliotetraosylceramide and gangliotriosylceramide) (Hartmann et al., Infect. Immun. 65: 1729-1733, 1997); Helicobacter pylori (gangliotetraosylceramide, gangliotriaosylceramide, sulfatides and GM3) (Huesca et al., Infect. and Immun. 64: 2643-2648, 1996; Kamisago et al., Infect. and Immun. 64:624-628, 1996; Simon et al., Infect Immun. 65: 750-757, 1997; Wadstrom et al., Curr. Microbiol. 34: 267-272, 1997); Borrelia burgdorferi (galactocerebroside; Virulent strain 297; glycosylceramide, lactosylceramide, and galactosylgloboside) (Garcia Monco et al., Neurology 42:1341-1348, 1992; Kaneda et al., Infect. and Immun. 65: 1138-1141, 1997); and Pseudomonas aeuroginosa and Candida albicans (asialo GM1) (Yu et al., Infect. and Immun. 62: 5213-5219, 1994).
Virus binding can be mediated via sphingolipids, including HIV-1 gp120 (galactosylceramide) (Fantini et al., J. Biol. Chem. 272: 7245-7252, 1997), Sendai virus (ganglioside GD1a) (Epand et al., Biochemistry 34: 1084-1089, 1995); and influenza viruses (gangliosides, sulfatides and polyglycosylceramides) (Fakih et al., Infect. and Immun. 65: 1695-1700, 1997; Matrosovich et al., Virology 233: 224-234, 1997; Matrosovich et al., Virology 223: 413-416, 1996; Sato et al., Biochim. Biophys. Acta 1285: 14-20, 1996; Suzuki et al., Biochem. J. 318:389-393, 1996).
Sphingolipids are significant components of foods. The types of sphingolipids differ among organisms (and for different types of cells within an organism); for examples, the predominant sphingolipid classes for plants are cerebrosides (Lynch, D. V., Lipid Metabolism in Plants (T. S. Moore, Jr., ed.), pp. 285-308, CRC Press, Boca Raton, Fla. 1993), ceramide phosphoinositols for yeast (Lester et al., Biochim. Biophys. Acta. 1165:314-320, 1993), and sphingomyelin (plus a wide spectrum of neutral and acidic glycolipids) for mammals (Hakomori, J. Biol. Chem. 265:18713-18716, 1983). There are also differences in the nature of the sphingoid-base backbones because plants contain predominantly 4-hydroxysphingenine (t18:1) isomers and 4,8-sphingadienine (d18:2) isomers (Lynch, D. V., Lipid Metabolism in Plants (T. S. Moore, Jr., ed.), pp. 285-308, CRC Press, Boca Raton, Fla. 1993), ceramide phosphoinositols for yeast (Lester et al., Biochim. Biophys. Acta., 1165:314-320, 1993). Furthermore, considerable structural diversity exists among the sphingoid base (and fatty acid) composition of glucocerebrosides isolated from different plant tissues. Sphingoid bases with a double bond at position 8 have been seen in mammalian sphingolipids (Karlsson, K. A., Chem. Phys. Lipids, 5:6-43, 1970), but have never been shown to be synthesized by mammalian cells; therefore, it is likely that they are acquired by mammals by consumption of plant sphingolipids.
Sphingolipids not only help define the structural properties of membranes, but also play important roles in cellxe2x80x94cell and cell-substratum interactions, and help regulate growth and differentiation by a variety of mechanisms, such as inhibition of growth factor receptor kinases and effects on numerous cellular signal transduction systems (for reviews see Hakomori, J. Biol. Chem. 265:18713-18716, 1991; Bell et al., Advances in Lipid Research: Sphingolipids and Their Metabolites, vols. 25 and 26, Academic Press, Sand Diego, 1993; Merrill and Sweeley, New Comprehensive Biochemistry: Biochemistry of Lipids, Lipoproteins, and Membranes (Vance, D. E. and Vance, J. E., eds.), 1996; Spiegel and Merrill, FASEB J 10, 1388-1397, 1996). The backbones of dietary sphingolipids (ceramides and sphingoid bases) are assumed to induce cellular responses that are normally regulated by intracellular sphingolipid second messengers. This may have implications far beyond just their effects on carcinogenesis, such as restenosis, where there is proliferation of smooth muscle cells in the course of development of plaques in vascular tissue, and closing off of the artery after angioplasty.
Interest in these lipid backbones as bioactive compounds began when Hannun, Loomis and Bell at Duke University serendipitously discovered that sphingosine is a potent inhibitor of protein kinase C in vitro (Hannun et al., J. Bio. Chem. 261:12604-12609, 1986), and by the finding that sphingoid bases also inhibit diverse cell functions requiring protein kinase C (Hannun et al., J. Bio. Chem. 261:12604-12609, 1986; Wilson et al., J. Biol. Chem. 261:12616-12623, 1986; Merrill et al., J. Biol. Chem. 261:12610-12615, 1986). Subsequent studies have found that sphingosine activates and inhibits several protein kinases, and affects a large number of signal transduction systems. Researchers thereafter uncovered additional bioactive sphingolipid xe2x80x9cbackbonesxe2x80x9d such as ceramides (Hannun, J. Biol. Chem. 269:3125-3128, 1994; Kolesnick, Cell 77:325-328, 1994), sphingosine 1-phosphate (Zhang et al., J. Cell Biol. 114:155-167, 1991; Spiegel et al., J. Lipid Mediators 8:169-175, 1993), and N-methyl-sphingosines (Igarashi, J. Biol. Chem. 265:5385-5389, 1990). As the cellular targets of these compounds have been studied, they have turned out to be as complex as for other lipid-signaling pathways in cells, i.e., they involve activation and inhibition of protein kinases, phosphoprotein phosphatases, ion transporters, and xe2x80x9ccross-talkxe2x80x9d with other signaling pathways.
The current paradigm for the action of sphingolipids in cell regulation is that complex sphingolipids are important in membrane structure, especially specialized membrane functions such as are found in calveolae. They also interact with cell surface receptors for growth factors, and the extracellular matrix. The lipid backbones (ceramide, sphingosine and sphingosine 1-phosphate) function as xe2x80x9csecond messengersxe2x80x9d to affect protein kinases, phosphoprotein phosphatases, ion transporters, and other regulatory machinery, as illustrated in FIG. 3. As examples, tumor necrosis factor-xcex1, interleukin 1xcex2, and nerve growth factor induce sphingomyelin hydrolysis to ceramide as a second messenger (Hannun, J. Biol. Chem. 269:3125-3128, 1994; Kolesnick, Cell 77:325-328, 1994); other agonists, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to sphingosine, and activate sphingosine kinase to form sphingosine 1-phosphate (Olivera and Speigel, Nature 365:557-560, 1993; Coroneos et al., J. Biol. Chem. 270:23305-23309, 1995). Depending on the cell type, these metabolites can either stimulate or inhibit growth. While the details of growth regulation by ceramide, sphingosine, and sphingosine 1-phosphate are still being uncovered (Spiegel and Merrill, FASEB J., 1996), depending on the system, it appears to involve calcium mobilization from intracellular stores, and activation of the MAP (and Jun) kinase pathways and transcription factors (AP1) (Su et al., J. Biol. Chem. 269:16512-16517, 1994), induction of retinoblastoma protein dephosphorylation (Pushkareva et al., Biochemistry, 34:1885-1892, 1995), and in some cases, induction of apoptosis (Obeid et al., Science, 259:1769-1771, 1993; Jarvis et al., Cancer Res. 54:1707-1714, 1994).
Carcinogenesis involves progressive genetic mutations leading to the loss of cell growth regulation and, as importantly, loss of regulation of programmed cell death (apoptosis). The genetic abnormalities have been studied extensively for colorectal cancer and include activation of oncogenes and loss of tumor suppressor genes as cells progress from early premalignant lesions to carcinomas (Fearon and Vogelstein, 1990; Jen et al., 1994; Kinzler and Vogelstein, 1996), and a progressive inhibition of apoptosis (Bedi et al., 1995).
It has been determined that free sphingoid bases and ceramide, through inhibition of protein kinase C (Hannun et al., J. Biol. Chem. 261:12604-12609, 1986; Stevens et al., Biochem. Biophys. Acta 1051:37-45, 1990a) or induction of retinoblastoma protein dephosphorylation (Pushkareva, Biochemistry 34:1885-1892, 1995; Obeid and Hannun, Trends Biochem. Sci. 20:73-77, 1995) can inhibit cell growth; sphingoid bases (Stevens et al., Cancer Res. 50:222-226, 1990b) and ceramide (Okazaki et al., J. Biol. Chem. 265:15823-15831, 1990) can promote cell differentiation; and, ceramide (Obeid et al., Science 259:1769-1771, 1993; Hannun and Obeid, Trends Biochem. Sci. 20:73-77, 1995; Kolesnick and Fuks, 1995) as well as sphingosine (Ohta et al., Cancer Res. 55:691-697, 1995) can induce apoptosis, apparently through suppression of Bcl-2 (Sakakura et al., FEBS Lett. 379:177-180, 1996; Chen et al., Cancer Res. 55:991-994, 1995). Wright et al. (FASEB J 10:325-332, 1996) have recently reported that U937 cells that were isolated based on their resistance to TNF-induced apoptosis have a defect in the activation of sphingomyelinase and the 24 kDa apoptotic protease (AP24); furthermore, this also makes the cells resistant to UV-induced apoptosis. This raises the possibility that some tumors could be defective in apoptosis because they have mutation(s) in upstream regulators of sphingomyelin turnover to ceramide. If so, addition of exogenous ceramide (and/or sphingosine or synthetic analogs of either) might bypass this (these) defects.
There have been few studies of the relevance of these compounds to carcinogenesis. Several years ago, it was explored whether free sphingoid bases could inhibit transformation using C3H 10T1/2 cells initiated by xcex3-irradiation or chemical carcinogens as a model system (Borek et al., Proc. Natl. Acad. Sci. USA 88:1953-1957, 1991; Borek and Merrill, Antimutagenesis and Anticarcinogenesis Mechanisms III (G. Bronzetti et al. eds., Plenum Publishing Corp., New York), 1993). These studies showed that sphingosine and sphinganine completely blocked xe2x80x9cpromotionxe2x80x9d of transformed foci by phorbol esters, and reduced the number of transformed foci in cells treated with just xcex3-irradiation. The sphingoid bases were added at levels that affected protein kinase C but were not noticeably cytotoxic; therefore, the effects were not due to non-specific killing by a lipid xe2x80x9cdetergent.xe2x80x9d
In another study, female CF1 mice (6 weeks of age, 10 mice per group) were fed a standard, defined diet (AIN76A) (without sphingolipid supplementation), then treated with 1,2-dimethylhydrazine (DMH) in 6 weekly injections (40 mg/kg). One week after the last DMH administration, some of the mice were changed to diets supplemented with 0.05% sphingomyelin, and analyzed on week 7 for aberrant colonic crypts (McLellan et al. Cancer Res. 51:5270-5274, 1991; Pretlow et al., Cancer Res. 51:1564-1567, 1991). Sphingomyelin caused a significant (50%) reduction in the number of aberrant colonic crypts induced by DMH (Dillehay et al., J. Nutr. 124:615-620, 1994). All of the aberrant crypts were found in the distal third of the colon.
Aberrant colonic crypt foci are one of the earliest morphological changes of colonic cells to dysplasia. It is thought that some of these lesions will eventually develop into adenomas and carcinomas (McLellan and Bird, Cancer Res. 51:5270-5274, 1991; Pretlow et al., Cancer Res. 51:1564-1567, 1991; Bruce et al., Mutat. Res. 290:111-118, 1993; Toribara and Sleisenber, 1995), which makes aberrant crypt foci a useful biomarker for studies of agents that may inhibit (or enhance) colon carcinogenesis. The effect of sphingomyelin on DMH-induced carcinogenesis was evaluated in a pilot experiment (15 mice per group) conducted with milk sphingomyelin, and a larger experiment (40 mice per group) with buttermilk sphingomyelin. Mice fed sphingomyelin has about half of the tumor incidence of the control. A control group fed sphingomyelin, but not treated with DMH, had not signs of a deleterious effect of consumption of sphingolipids, and developed no tumors.
A second study with buttermilk sphingomyelin gave a somewhat different, but nonetheless very interesting result. There was no reduction in the number of overall tumors, but histological analyses revealed that mice fed sphingomyelin had a shift in tumor type such that there were more adenomas versus adenocarcinomas than for the control (defined as polypoid adenomas by the presence of an intact muscularis mucosa versus invasion of neoplastic cells through the muscularis mucosa, which is characteristic of adenocarcinomas).
The first studies of sphingolipid digestion were published by Nilsson approximately 30 years ago (Nilsson, A., Biochim. Biophy. Acta. 164:575-584, 1968; Biochim. Biophys. Acta 187: 113-121, 1969) and indicated that sphingomyelin was metabolized in the small intestine and up to 30% of the radiolabel could be recovered in lymph. However, 21 to 46% of the radiolabel was found in feces, mainly as ceramide, which indicated that the digestion of sphingomyelin is not very efficient, but may continue in the lower bowel (probably with the involvement of intestinal microflora). Secondly, the time course for the absorption of radiolabel was somewhat curious, with peak absorption 10 hours after feeding the sphingolipid, which may indicate that coprophagy had occurred. A similar study with the glycolipids glucosylceramide and galactosylceramide (Nilsson, Biochim. Biophy. Acta. 164: 575-584, 1969) found that glycolipids are also only partially digested.
Follow-up studies of sphingolipid digestion by rats and mice were conducted using several approaches (Schmelz et al., Journal of Nutrition, 124: 702-712, 1994). The first is a simpler model, the use of isolated segments of the intestine in which the animal is anesthetized and the intestines are flushed, tied-off, and then injected with the radio labeled compound of interest. The advantage of this technique is that the intestine retains most of its normal blood supply, and nutrient absorption can be fairly simply assessed by the disappearance of the radiolabel (or appearance in the tissue or blood). When [3H-sphingosyl]sphingomyelin was added to segments of the jejunum, ileum, and caecum, or colon, very little radiolabel was lost from the segments; radiolabel became associated with the tissue in a form that was not removed by washing. It was found that the [3H-sphingosyl]backbone had been incorporated into other classes of sphingolipids. This established that the sphingolipid had undergone some hydrolysis, cellular uptake and metabolism. It is not known whether hydrolysis occurred before or after cellular uptake, and subsequent double labeling experiments have indicated that both the [3H-sphingosyl]backbone and [14C] choline head group are taken up.
In a second approach (Schmelz et al., Journal of Nutrition. 124; 702-712, 1994), [3H-sphingosyl]-sphingomyelin was given to mice by gavage, and then the animals were killed after different time intervals and the amount of radiolabel in different intestinal segments measured. From 3 to 5% of the radiolabel had progressed into the colon within about 2 hours, which confirmed the earlier observation by Nilsson (Biochim. Biophys. Acta., 164: 575-574, 1968) that at least some sphingomyelin escapes digestion by the upper intestine and arrives in the colon. About 5% of the label appeared in liver, sphingolipids, so there is some uptake of the sphingoid base from the diet, and incorporation into the tissues of the animal. The lack of an effect on weight gain was important because carcinogenesis studies would be more difficult to interpret if sphingolipids had affected growth (Birt et al., 1992).
Current knowledge about sphingolipids and the recent findings summarized above establish four points. First, sphingolipids, and especially sphingosine and ceramide, are highly bioactive compounds with potential to serve as naturally occurring modulators of diverse cell behaviours that include neoplastic transformation. Second, findings in various cell culture systems and animal models for carcinogenesis suggest strongly that sphingolipids suppress carcinogenesis. Third, glycosphingolipids may be more effective than phosphosphingolipids because when they are hydrolyzed to a lysosphingolipid (such as psychosine for cerebrosides) the product is also cytotoxic, in contrast to lysosphingomyelin, which is mitogenic. As a result, they may be selectively cleaved in the lower GI track. Fourth, based on the above-described dietary studies, only a small amount of orally administered sphingolipid survives to the lower intestine, and logically, to other distant sites of the body which might be in need of treatment.
U.S. Pat. Nos. 5,232,837 and 5,518,147 issued to Merrill, Wong, and Riley disclose a method of altering the metabolism of sphingolipids in a cell comprising exposure of the cell to fumonisins, or an analog thereof. Fumonisins (see FIGS. 5 and 6) are a family of mycotoxins produced by Fusarium moniliforme and related fungi that are common contaminants of maize, sorgum, and related grains. Fumonisins and sphingosine share a 2-amino-3-ol head group and possess backbone carbon chains of approximately equal size. However, sphingosine is hydroxyl-substituted at carbon 1 while fumonisin B1 and B2 are hydroxyl-substituted at carbon 5. In addition, the fumonisins possess polycarboxylate moieties attached to the tail of the backbone carbon chain. Similarities between sphingosine and Fumonisin in their head groups and tails are responsible for the activity of the fumonisins. It was discovered that fumonisin acts as an inhibitor of ceramide synthase, and thus alters the metabolism of the conversion of sphinganine to dihydroceramide.
An abstract published in J. Urol., 155(5):653A, Abst. 1367, 1996, disclosed that the administration of fumonisin in combination with doxirubicin exhibited a striking ability to treat renal cell carcinoma. This abstract showed that Fumonisin B has cytostatic activity using the Soft Agar thymidine incorporation in vitro assay model of human carcinomas against renal bladder and colon cancer cell lines. It confirmed that Fumonisin B can augment the cytotoxicity of other cytotoxic agents in this model system.
U.S. Pat. No. 5,190,876 and U.S. Pat. No. 5,459,057 to Merrill, Kinkade, and Stevens, disclose a method and composition for enhancing the action of biological response modifiers, such as promoting cellular differentiation, by administration of an effective amount of Vitamin A or an analog thereof with sphingosine or an analog thereof.
U.S. Pat. No. 5,110,987 to Liotta and Merrill discloses a method for preparing sphingosine derivatives.
U.S. Pat. No. 5,635,536 to Lyons discloses emulsions suitable for administration of sphingolipids.
In light of the fact that sphingolipids play a fundamental role in a number of metabolic pathways, including cell proliferation and programmed cell death, it would be of benefit to provide new sphingolipid derivatives that have improved properties, bioavailability, or are targeted to desired locations for effective therapy.
It is therefore an object of the present invention to provide new sphingolipids with biological activity, and in particular, sphingolipids that are useful in the treatment of abnormal cell proliferation, including benign and malignant tumors, the promotion of cell differentiation, the induction of apoptosis, the inhibition of protein kinase C activity, the modification of the colonization of microfora in the body, and the treatment of inflammatory conditions including psoriasis, inflammatory bowel disease as well as proliferation of smooth muscle cells in the course of development of plaques in vascular tissue.
It is therefore an object of the present invention to provide sphingolipids in a prodrug form that can be cleaved to a parent sphingolipid in vivo to increase bioavailability and or efficacy. It is another object of the present invention to provide sphingolipids in a manner that efficiently targets them to a desired location.
It is still another object of the present invention to provide a method and composition that increases the level of sphingolipid that is delivered to a desired location in a host animal.
It is a further object of the present invention to provide new sphingolipid compositions for the treatment of cancer, including colon cancer.
It is yet another object of the present invention to provide new methods for the treatment of bacterial infections.
It is still another object of the present invention to provide new sphingolipid compositions for the modification of the colonization of microfora that influence colon cancer and other intestinal disorders.
Derivatives of sphingolipids of the formula: 
are provided wherein:
A is a spacer group which is (CH2)m where m=0-14, where any of the hydrogens may be independently replaced by R1 or X and where any two adjacent carbons may be independently replaced by a C3-C8 cycloalkyl group, a 1,2-, 1,3- or 1,4-disubstituted benzene group, or a 2,3-, 2,4- or 2,5-disubstituted thiophene, furan or pyrrole group;
X, Y, V, and Q are independently hydrogen, OR1, NR2, CN, alkyl, acyl (i.e., C(O)R), carboxylate (i.e., OC(O)R), and wherein alternatively, V and Y, Y and Q or Q and A can together constitute a double or triple bond;
W=no substituent, H, alkyl, aryl, alkenyl, alkynyl, alkaryl, aralkyl, C(O)(CH2)nCO2H, C(O)(CH2)nCWxe2x80x22CO2H, or OR1;
Wxe2x80x2 is selected independently from H, alkyl, aryl, (CH2)nCO2H; (CH2)nxe2x80x99l CH(CO2H)CH2CO2H; and (CH2)nCH(CO2H)CH(CH2CO2H)CO2H;
Z is H, O, NH, NR, NHC(O), C(O)OR1, C(O)NH, or C(O)NR;
R is selected independently from H, alkyl, acyl, alkenyl, alkynyl, aryl, alkaryl, aralkyl, or heteroaryl;
R1 is R or R2;
R2 is phosphate (OP(OR3), wherein at least one R is not hydrogen), b-D-galactoside, N-acetyl-b-D-glucosamine, a-D-mannoside, an organic azo-bond containing moiety that can be reduced by an azoreductase, b-D-cellobiosides, b-D-glucopyranosides, b-D-galactopyranosides, b-D-glucuronides, starch (such as maize starch, amylo-maize starch, pectin and others found in wheat flour, potato and beans), lactose, raffinose, stachyose, fructooligosaccharides (such as oligofructose and inulin), an amide or ester of b-cyclodextrin, dextran linked via succinate and glutarate, an amino acid or peptide, or a polyamino acid or polypeptide, furanose and pyranose carbohydrates, sulfonate (and esters thereof), phosphocholine, phosphoserine, and phosphoethanolamine;
wherein there is at least one R2 substituent in the sphingolipid derivative.
It has been discovered that biologically important sphingolipids can be administered as prodrugs which increase the level of active compound that is delivered to the active site of interest. The prodrug is cleaved by an appropriate enzyme in vivo to release a parent sphingolipid moiety for desired therapy. Certain derivatives of Formula I are especially suited for treatment of disorders of the lower intestinal tract, including but not limited to colon cancer, intestinal polyps, intestinal tumors, inflammatory bowel diseases including ulcerative colitis and Crohn""s disease, necrotizing enterocolitis, and ileocecitis, other inflammations of the lower bowel, and antibiotic associated colitis, as the enzymes which free the sphingolipid from its prodrug form are concentrated in the lower intestine. These prodrugs are resistant to hydrolysis in the upper gastrointestinal tract, and are more readily cleaved in the cecum and the colon. This invention thus addresses the problem to date of low bioavailability of these important compounds at the target site.
In another alternative embodiment of the invention, R2 is a xe2x80x9ctargeting moietyxe2x80x9d that binds to a receptor molecule on the target membrane""s surface. Examples of targeting molecules include steroids, hormones (including but not limited to melanin), hormone receptors, cell specific receptors and ligands that bind to cell specific receptors (including but not limited to sugars, proteins, peptides, and glycoproteins), antibodies (for example, the Her 2-Nu antibody for the treatment of breast cancer), antibody fragments (such as the Fab or Fab2 antibody fragments), antigens, T-cell receptor fragments including T-cell receptor variable regions. In one embodiment, a steroid that binds to the membrane of a cancer cell is used as the targeting moiety.
The active compounds are administered in an effective amount to alter sphingolipid metabolism, and thus are useful in the treatment of abnormal cell proliferation, including tumors, cancer, psoriasis, and familial polyposis; in the promotion of cell differentiation, in the induction of apoptosis, in the inhibition of protein kinase C; and in the modification of the colonization of microflora that influence colon cancer and other intestinal disorders, and in the treatment of inflammatory conditions. These compounds either exhibit activity in themselves or act as a prodrug of a parent compound. The compounds can be administered alone or in combination with other biologically active compounds to achieve the desired effect.
These compounds can in particular be used to treat infections caused by bacteria (gram negative and gram positive) and viruses which have receptors for sphingolipids as anchoring means for colonization. Nonlimiting examples of microorganisms that can be treated using this method include cholera toxin, verotoxin, Shiga-like toxin 2e, Clostridium botulinum type B neurotoxin, Escherichia coli, Haemophilus influenzae; Helicobacter pylori; Borrelia burgdorferi, Pseudomonas aeruginosa, Candida albicans, HIV, Sendai virus, and influenza viruses. In this embodiment, an effective amount of a selected compound falling within the above formula is administered to decrease or prevent colonization of the selected microorganism.
In yet another embodiment of the invention, a derivative of a fumonisin is provided that includes at least one covalently bound R2 group. These compounds are also useful in the treatment of abnormal cell hyperproliferation, including cancer and tumors, most notable when used in combination with other antitumor compounds. In particular, the derivatives can be used to target cells, including solid tumors such as colon or urogenital tract tumors including kidney, bladder, prostate, as well as the uterus and cervix, for enhanced therapy.
In yet another embodiment of the invention, sphingolipid derivatives are also useful for the modification of the colonization of microfora that influence colon cancer and other intestinal disorders.
It has also now been discovered that sphingoid bases (including naturally occurring compounds that share this structural motif, such as the fumonisins), including but not limited to those in Formula I and in FIGS. 1-5 trigger the release of cytochrome c from mitochondria into the cytosol, resulting in the cleavage and activity of caspase-3 and thereafter, apoptosis. These compounds are thus useful in the treatment of any disorder which is mediated by, or benefited by, regulation of cell growth, differentiation, and/or induction of cell death. In addition, treatment with sphingolipids has been shown to down-regulate Bcl-2 levels, which further sensitizes cancer cells to apoptosis induced by other apoptotic stimuli including chemotherapeutic drugs, xcex3-radiation and Fas ligand. Therefore, treatment with sphingolipids sensitizes cancer cells simultaneously by promoting the cytosolic accumulation of cytochrome c and by down-regulating Bcl-2, which acts as a barrier to this event.
It has been discovered that compounds of the above formula can be used to treat bacterial infections.
It is still another object of the present invention to provide new sphingolipid compositions for the modification of the colonization of microfora that influence colon cancer and other intestinal disorders.